Acquired immune deficiency syndrome (AIDS) has been recently recognized in several countries. Due to its devastating effect on the patients and indications that the disease is spreading, much effort has been devoted to elucidate and identify the cause of the disease. Epidemiologic data suggests that AIDS is caused by an infectious agent that is horizontally transmitted by intimate contact or exposure to blood or certain blood products.
In 1983, F. Barre-Sinoussi et al. of the Institute Pasteur reported the isolation of a T-lymphotropic retrovirus from a patient at risk for AIDS. The retrovirus appeared to be a member of the human T-cell leukemia virus (HTLV) family. However, its immunological response is distinct from known HTLV-I or HTLV-II. F. Barre-Sinoussi et al., Science, 220, pp. 868 (May, 1983).
A similar virus, designated HTLV-III, has also been isolated by R. C. Gallo's group at National Cancer Institute from the blood samples of a large number of AIDS and ARC patients by co-cultivation with a permissive T-cell line H9. See Popovic, M. et al., Science, 224, pp. 497 (1984) and Gallo, R. et al., Science, 224, pp. 500 (1984).
V. S. Kalyanaraman et al. of the Center for Disease Control, Atlanta, Ga., reported the isolation of a lymphadenopathy associated virus (LAV) in patients with AIDS and the development of a radioimmuno-precipitation assay using the major core protein, p25, of LAV. Their test procedure involved the use of the LAV virus propagated in primary cultures of blood lymphocytes and harvested. The p25 core protein was isolated from the harvested virus, labelled with I.sup.125 and used as target antigen. The labelled antigen was added to serum and precipitation of at least 15% of the labelled antigen is regarded as a positive result. See V. S. Kalyanaraman et al., Science, 225, pp. 321 (July 1984). However, based on the reported results, the test was positive only for 41% of the AIDS patients and 72% positive for patients with lymphadenopathy syndrome (LAS) otherwise known as ARC. This means that the procedure is not sufficiently sensitive or accurate to be used as a detection or diagnostic tool for screening serum for the presence of antibodies to the AIDS virus.
LAV and HTLV-III, as well as various strains of related retrovirus isolated from AIDS patients share several important characteristics. See Feorino, P. et al., Science, 225, pp. 69 (1984); Levy, J. et al., Science, 225, pp. 840 (1984). These include viral replication in OKT4.sup.+ human T-cell leukocytes, in vivo and in vitro; association with impaired T-cell proliferation, the appearance of cytopathic effects; (See Montagnier et al., Human T Cell Leukemia Virus, pp. 363, Cold Spring Harbor Laboratory, 1984; Popovic, M. et al., op. cit., and Klatzmann, D. et al. Science, 225, pp. 59 (1984)) and recognition by antibodies in the sera of AIDS and ARC patients. See Montagnier, et al., op. cit.; Levy J. et al., op. cit., Sarngadharan, M. et al., Science, 224, pp. 505 (1984); Safai, B. et al., Lancet, i, pp. 1438 (1984); Brun-Vezinet, F., et al., Lancet, i, pp. 1253 (1984); Brun-Vezinet, F. et al., Science, 226, pp. 453 (1984); Goldbert, J. et al., Lancet, ii, pp. 711 (1984) and Laurence J. et al., New England J. Med., 311, pp. 1269 (November 1984).
In November 1984, L. W. Kitchen et al. reported the use of a HTLV-III infected line, designated H9/HTLV-III, to test the incidents of AIDS in haemophiliac patients. The method involved inactivation of the virus with 2% paraformaldehyde in phosphate buffer and used of the inactivated cells to determine if haemophiliac patients have been inadvertently exposed to AIDS virus through blood transfusion. The data using sera samples from 50 haemophiliacs show that there is an increasing risk for these patients to contract AIDS, because of their need for blood transfusions to sustain life. L. W. Kitchen et al., Nature, 312, pp. 367 (November 1984). This means that there is an urgent need for a safe, reliable and sensitive test to screen each blood sample before its inclusion in blood banks to isolate blood samples which have been contaminated with AIDS virus, and thus avoid the inadvertent spread of AIDS among patients who must receive blood transfusions, e.g. haemophiliac and surgical patients.
In November 1984 and January 1985, R. C. Gallo's group at National Cancer Institute and other collaborators positively concluded that HTLV-III is the causative agent of AIDS and reported the nucleotide sequence of HTLV-III. See Beatrice Hahn et al., Nature, 312, pp. 166 (November 1984), George M. Shaw et al., Science, 226, pp. 1165 (December 1984) and Lee Ratner et al., Nature, 313, pp. 277 (January 1985).
Meanwhile, three other groups also reported the complete nucleotide sequence of the AIDS virus. See Muesing et al., Nature, 313, pp. 450 (February 1985); Sanchez-Pescados, R. et al., Science, 227, pp. 484 (February 1985) and Wain-Hobson et al., Cell, 40, pp. 9 (January 1985). These reports elucidated the structure of the HTLV-III virus at both the DNA level and the projected protein level and permit further serological studies of the different epitopes present on the HTLV-III virus.
Simultaneously, the group at Institute Pasteur reported that LAV has been identified as a causative agent for AIDS, and is considered to be identical to HTLV-III. The assay procedure used by this group also involves propagating LAV in T4.sup.+ cells of healthy individuals. The viral antigen was then concentrated and deactivated in 0.5 percent sodium dodecyl sulfate at 37.degree. C. for 15 minutes. Serum samples were then tested against the antigen in an enzyme immunoassay with orthophenylene diamine as substrate. The presence of antibody in serum was found in 68% of AIDS patients, 100% of patients with Kaposi's sarcoma and 100% of pre-AIDS patients. Jeffrey Laurence et al., op. cit.
Recently, U.S. Pat. No. 4,520,113 was issued to R. C. Gallo et al. The Gallo et al. patient describes a method of detecting antibodies in sera of AIDS and pre-AIDS patients by using lysates of a cell line, designated H9/HTLV-III, as the antigen in an enzyme-linked immunosorbent assay (ELISA) or in a strip radioimmunoassay based on the Western Blot technique or an indirect immunofluorescent method. The method is about 85% accurate. The Gallo patent further indicated that several antigens from HTLV-III, p65, (MW 65,000), p60 (MW 60,000), p55 (MW 55,000), p24 (MW 24,000) and p41 (MW 41,000) are recognized by antibodies in sera from AIDS patients, homosexuals and heroin addicts. Of these, major immune reactivity or specificity is directed against p41, a protein constituting the envelope antigen of HTLV-III. This patent further states that it is believed that additional purification and refinement of p41 may lead to an even more sensitive ELISA assay. Based on this statement, the antigen suitable as a test reagent is to be a p41 segment derived from HTLV-III cultivated in H9 cell line.
It is further reported in Robert C. Gallo et al., Science, 228, pp. 93 (April 1985) that a combined cloning and expression system in E. coli has been used to identify HTLV-III encoded peptides which react immunologically with antibodies in sera from AIDS patients. Closed HTLV-III DNA was sheared into fragments and inserted into an expression vector. The inserted DNA was then expressed in E. coli transformants. Of 300 clones tested, 20 showed specific reactivity with sera from AIDS patients. The 20 clones were analyzed and found to contain segments from the ORF segment of HTLV-III and were identified as clones 175, 191, 13, 31, 162, 113, 121 and 127. Of the eight clones, ORF clones 113, 121 and 127 define the protein encoded by the portion of the env-lor region produced by HTLV-III infected cells and induces antibody production in most if not all AIDS patients.
All of the reported assay procedures for detecting antibodies to HTLV-III and for diagnosis of AIDS or pre-AIDS conditions involve the use of the HTLV-III or LAV virus. None of the procedures are 100% accurate. This is undesirable for use in the screening of sera in blood banks. The less than 100% accuracy of the tests may permit contaminated sera from escaping detection and be used in blood transfusions to the severe detriment of blood recipients. Moreover, the use of the HTLV-III virus as the testing agent is dangerous to healthy laboratory workers, requiring extreme precautions to avoid all chances of exposure during the preparative process to make the test reagent. Furthermore, even though the deactivated virus is used in some of the published procedures, exposure to the deactivated virus can cause antibody production in healthy workers, who may then be falsely diagnosed as having AIDS, ARC or pre-AIDS condition. Moreover, presence of cellular materials from H9 cells or E. coli in the test agent may elicit a false positive response in the HTLV-III antibodies screening test from individuals who have antibodies to E. coli or H9 cells. These false positive reactions can bring severe anxiety to the healthy individuals and their family and may lead to a healthy individual being mistakenly diagnosed as having AIDS and be exiled from normal social activities as a consequence.
Furthermore, up to the present, no viable vaccine or method to provide protection against HTLV-III has been reported for AIDS, ARC or pre-AIDS conditions. The use of deactivated virus provokes fears of contracting the disease and would prevent its acceptability and use.
Similarly, the development of monoclonal and polyclonal antibodies to HTLV-III in mammals involves the use of HTLV-III as the immunogen and this presents similar risks in the procedure.
It is, therefore, an objective of the present invention to develop a detection or diagnostic procedure that does not require the use of the virus or lysates thereof as a test reagent.
A further objective is to develop a test procedure that is highly sensitive and accurate, preferably 100% accurate.
Another objective is to develop a test that is highly sensitive so that very little test reagent or body fluid is needed to obtain an accurate result.
A further objective is to prepare a test reagent by chemical means, which test reagent can be used to detect the presence of antibodies to HTLV-III in body fluids and diagnose AIDS, ARC or pre-AIDS conditions, thereby avoiding the danger of exposure to the virus or segments thereof and the unnecessary proliferation of the virus.
Another objective is to develop a vaccine which when introduced into the body will stimulate production of antibodies to HTLV-III to provide protection against infection by HTLV-III or LAV in healthy mammals, including humans.
A further objective is to provide an immunogen which can be used for the development in mammals of monoclonal and polyclonal antibodies to HTLV-III which does not involve the use of HTLV-III as the immunogen.